Luis A. Martinez and Aras Petrulis Neuroscience Institute
Georgia State University, Atlanta, Georgia, USA 30302
Previously published in Hormones and Behavior 63(4): 606-‐614
4.1 Abstract
Precopulatory behaviors that are preferentially directed towards opposite-‐sex conspecifics are critical for successful reproduction, particularly in species wherein the sexes live in isolation, such as Syr-‐ ian hamsters (Mesocricetus auratus). In females, these behaviors include sexual odor preference and vaginal scent marking. The neural regulation of precopulatory behaviors is thought to involve a network of forebrain areas that includes the medial amygdala (MA), the bed nucleus of the stria terminalis (BNST), and the medial preoptic area (MPOA). Although MA and BNST are necessary for sexual odor preference and preferential vaginal marking to male odors, respectively, the role of MPOA in odor-‐ guided female precopulatory behaviors is not well understood. To address this issue, female Syrian hamsters with bilateral, excitotoxic lesions of MPOA (MPOA-‐X) or sham lesions (SHAM) were tested for sexual odor investigation, scent marking, and lordosis. MPOA-‐X females did not investigate male odors more than female odors in an odor preference test, indicating that MPOA may be necessary for normal sexual odor preference in female hamsters. This loss of preference cannot be attributed to a sensory deficit, since MPOA-‐X females successfully discriminated male odors from female odors during an odor
discrimination test. Surprisingly, no deficits in vaginal scent marking were observed in MPOA-‐X females, although these females did exhibit decreased overall levels of flank marking compared to SHAM fe-‐ males. Finally, all MPOA-‐X females exhibited lordosis appropriately. These results suggest that MPOA plays a critical role in the neural regulation of certain aspects of odor-‐guided precopulatory behaviors in female Syrian hamsters.
4.2 Introduction
Precopulatory behaviors that aid in the identification and localization of potential mating part-‐ ners are an important component of reproductive behavior for most mammals (Beach, 1976). For spe-‐ cies typified by non-‐cohabitating sexes such as Syrian hamsters (Mesocricetus auratus), these behaviors are essential for successful mating (Gattermann et al., 2001; Pfaff et al., 2008). Female Syrian hamsters engage in a number of different precopulatory or solicitational behaviors, including vaginal marking (a stereotyped scent marking behavior resulting in deposition of vaginal secretion) and preferential ap-‐ proach towards, and investigation of, opposite-‐sex odors (Petrulis, 2009). Although both vaginal marking and opposite-‐sex odor preference are behavioral responses that are preferentially directed towards male compared to female odors (Johnston, 1977; Martinez and Petrulis, 2011; Martinez et al., 2010; Petrulis and Johnston, 1999; Petrulis et al., 1999), odor preference is more clearly linked to the identifi-‐ cation and localization of potential mating partners, whereas vaginal marking plays a key role in attract-‐ ing mates. Indeed, the deposited secretion is highly attractive to male hamsters (Johnston and Schmidt, 1979; Johnston, 1974; Kwan and Johnston, 1980), and females deposit these marks in such a way as to direct the male to her nesting area (Lisk et al., 1983).
The expression of both sexual odor preference and vaginal marking depends critically on an in-‐ terconnected set of brain areas that are more broadly involved in processing conspecific odor infor-‐ mation (Petrulis, 2009). These areas include the medial amygdala (MA), the bed nucleus of the stria terminalis (BNST), and the medial preoptic area (MPOA) (Wood, 1998). Odor information detected by
the main and accessory olfactory systems is initially processed by MA and relayed to MPOA, either di-‐ rectly or via BNST (Coolen and Wood, 1998; Wood and Swann, 2005). Not surprisingly, neurons in these brain areas exhibit selective activation to opposite-‐ vs. same-‐sex odors in both male and female ham-‐ sters (DelBarco-‐Trillo et al., 2009; Maras and Petrulis, 2010a). These areas also appear to play specific, functional roles in female precopulatory behaviors. For example, lesions of MA eliminate preferential investigation of male vs. female odors and reduce overall levels of vaginal marking by female hamsters, but do not eliminate preferential vaginal marking in response to male odors (Petrulis and Johnston, 1999). In contrast, females with lesions of BNST do not vaginal mark preferentially to male odors, but do display a normal preference to investigate male odors more than female odors (Martinez and Petrulis, 2011). These data suggest that although BNST may be a critical component of the neural circuit regulat-‐ ing vaginal marking responses to sexual odors, it is not necessary for the expression of sexual odor pref-‐ erence; therefore, odor information relevant for sexual odor preference processed by MA likely bypass-‐ es BNST and continues to other limbic/hypothalamic areas connected to MA, such as MPOA.
Although there is substantial evidence suggesting MPOA is broadly involved in regulating sexual behavior in rodents (Hull and Dominguez, 2007; Sakuma, 2008), its specific role in odor-‐guided female precopulatory behaviors is not clear. In rats, radiofrequency lesions of MPOA decrease the amount of solicitational behaviors towards, and time spent with, a sexually-‐experienced male, and disrupt females’ preference for intact compared to castrated male rat odors (Xiao et al., 2005). Furthermore, excitotoxic lesions of MPOA decrease the preference of female rats to spend time with intact males compared to estrous females (Guarraci and Clark, 2006). However, it should be noted that other researchers have found no effects of electrolytic lesions of MPOA on sexual odor/partner preference in female rats (Paredes et al., 1998) or ferrets (Robarts and Baum, 2007). Although comparable data for the role of MPOA in sexual odor preference in female hamsters is not available, this area does appear to be in-‐ volved in other precopulatory behaviors that can be induced by opposite-‐sex odors. Indeed, large elec-‐
trolytic lesions of MPOA that also damaged BNST decrease vaginal marking during interactions with males (Malsbury et al., 1977), and radiofrequency lesions of MPOA decrease ultrasonic vocalizations by females following exposure to male hamsters (Floody, 1989).
A significant limitation of previous studies examining the role of MPOA in odor-‐guided precopu-‐ latory behaviors is the lack of specificity in disrupting MPOA vs. nearby areas such as BNST. This is a criti-‐ cal issue given that these areas are highly interconnected and share similar patterns of connectivity with other brain areas that regulate precopulatory behaviors (Coolen and Wood, 1998; Simerly and Swanson, 1986, 1988; Wood and Swann, 2005). As mentioned above, we have recently utilized discrete, excitotox-‐ ic lesions in order to determine the role of BNST in preferential vaginal marking and sexual odor investi-‐ gation (Martinez and Petrulis, 2011); however, it is not known if MPOA plays either a comparable or dis-‐ sociable role from that of BNST in the regulation of these behaviors. In order to address this issue, we administered excitotoxic lesions of MPOA to female Syrian hamsters and measured effects of lesions on sexual odor investigation and scent-‐marking responses. Given that specific lesions of MPOA disrupt sex-‐ ual odor preference in male hamsters (Been and Petrulis, 2010b), we hypothesized that MPOA would be necessary for the normal expression of preferential investigation of male odors by females. Further-‐ more, given the previously observed effects of MPOA/BNST disruption (Malsbury et al., 1977), and con-‐ sidering that implants of estradiol specifically into MPOA increase the expression of vaginal marking (Takahashi and Lisk, 1987; Takahashi et al., 1985), we hypothesized that MPOA would also be necessary for maintaining overall levels of vaginal marking.
4.3 Methods
Overview of design
Subjects were initially screened for adequate levels of vaginal marking to male odors (> 5 marks/10 min), and then received either bilateral, excitotoxic lesions of MPOA or sham surgeries. Fol-‐
lowing recovery, subjects underwent a series of behavioral tests. First, subjects were tested for their investigatory responses to male and female odors (Odor investigation tests). This consisted of an initial test to familiarize subjects with the testing apparatus (Clean test), followed by a volatile odor preference test utilizing conspecific odor stimuli (Preference test). Second, subjects were tested for their ability to discriminate the sexual identity of odor stimuli using a habituation-‐discrimination task (Odor discrimina-‐ tion test). Subjects were then tested for scent-‐marking responses to male or female stimuli on two days of the estrous cycle, diestrous day 2 and proestrus. Finally, to verify that MPOA lesions had not disrupt-‐ ed the ability of females to display copulatory behavior, subjects were tested during behavioral estrus for receptive sexual responses to a sexually experienced male.
Subjects
Adult female Syrian hamsters (Mesocricetus auratus) were purchased from Harlan Laboratories (Indianapolis, IL, USA) at approximately 7-‐9 weeks of age. In addition to experimental subjects, a sepa-‐ rate group of unrelated adult male and female Syrian hamsters was purchased from Harlan Laboratories to serve as stimulus animals. Animals were either individually housed (experimental subjects) or group housed (3-‐4 same-‐sex animals per cage; stimulus animals) in solid-‐bottom polycarbonate cages contain-‐ ing corncob bedding and cotton nesting material (Nestlets; Ancare, Bellmore, NY). Subjects and stimulus animals were maintained on a reversed light cycle (14:10 light:dark; lights out at 10 am), with all behav-‐ ior testing occurring during the first four hours of the dark portion of the light cycle. Food and water were available ad libitum. Animal procedures were carried out in accordance with the Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-‐23; revised 1996) and approved by the Georgia State University Institutional Animal Care and Use Committee. It should be noted that none of the sur-‐ vivable manipulations utilized in the present study resulted in animal mortality; furthermore, that all efforts were made to minimize the total number of animals used and their suffering.
Estrous cycle monitoring
Prior to screening for sufficient vaginal marking levels, subjects were examined daily for eight consecutive days in order to determine their stage of the estrous cycle. Subjects were gently restrained while vaginal secretion was manually extruded using a disposable probe, and the consistency of the se-‐ cretion was examined for stringy consistency indicative of behavioral estrus (Orsini, 1961). Once the day of behavioral estrus was identified, the two cycle days prior to estrus were defined as diestrous day 2 and proestrus, respectively (Johnston, 1977). Estrous cycles were also monitored for eight days follow-‐ ing surgery to ensure that this procedure had not disrupted cyclicity. Finally, in order to verify that es-‐ trous cycle stage had been properly inferred from cyclic changes in vaginal secretion consistency, fe-‐ males were tested for sexual receptivity in response to a male prior to the conclusion of behavioral test-‐ ing (see Lordosis test below). In all cases, ‘day’ refers to the dark phase of the light cycle.
Surgery
At two to three months of age, subjects were assigned to either a MPOA lesion group (MPOA-‐X) or a sham lesion group (SHAM). A matched random assignment procedure was used, such that initial levels of vaginal marking in response to male odors on proestrus were equivalent across subjects as-‐ signed to the MPOA-‐X and SHAM groups (see below). Subjects were first anesthetized with 2-‐3% isoflu-‐ rane gas (Baxter, Deerfield, IL) in an oxygen (70%) and nitrous oxide (30%) mixture, and then placed within a stereotaxic apparatus (Kopf Instruments, Tujunga, CA) with ear-‐ and incisor-‐bars positioned such that the top of the skull was level. Following a midline scalp incision, the skin and underlying tem-‐ poral muscles were retracted to expose the skull. A hand-‐operated drill was then used to make holes in the skull in order to expose dura. For MPOA-‐X subjects, the excitotoxin N-‐methyl-‐D-‐aspartic acid (20 mg/ml, 25 nl per injection site; Sigma, St. Louis, MO) was injected bilaterally via a Hamilton microinjec-‐ tion syringe (701R 10 μl syringe; Hamilton, Reno, NV) under stereotaxic control. A single injection of ex-‐ citotoxin was made per hemisphere, using the following coordinates: Anterior-‐posterior, +1.7 mm (rela-‐
tive to bregma); medial-‐lateral, ±0.6 mm (relative to bregma); dorsal-‐ventral, -‐7.4 mm (relative to dura) using published anatomical plates of the Syrian hamster brain (Morin and Wood, 2001). The excitotoxin was expelled over the course of one minute, and the syringe needle was then left in place for an addi-‐ tional nine minutes to allow sufficient time for the injection volume to disperse from the syringe tip. Sham surgeries were conducted in a similar manner to lesion surgeries except that no liquid was infused into the target sites.
Immediately prior to completion of all surgeries, the skull holes were sealed with bone wax and the incision closed with wound staples. Subjects were then injected with an analgesic agent (5 mg/kg; Ketofen, Fort Dodge Animal Health, Fort Dodge, IA). All subjects were allowed to recover for at least 14 days prior to post-‐operative behavioral testing.
Scent-‐marking tests
Odor stimulus and apparatus
Stimuli for scent-‐marking tests consisted of vacated cages (43 x 22 x 20 cm) previously occupied by male or female stimulus animals for four days. Estrous cycles of stimulus females were not moni-‐ tored; however, given that each cage contained at least three females, and the occupation period com-‐ prised the complete four-‐day estrous cycle, it is likely that female cage stimuli was a composite of multi-‐ ple cycle days. Approximately one to four hours prior to use, a researcher blind to the experimental condition of subjects prepared the stimulus cages as follows: Stimulus animals were removed from their cages along with any food pellets and caked urine present in the corncob bedding, the soiled cotton nesting material was distributed evenly across the bottom of the cage, and a 40 cm x 19 cm x 0.4 cm perforated Plexiglas plate was placed over the bedding just prior to testing. This plate was centered within the cage such that bedding within three cm of the outer walls of the stimulus cages remained directly accessible to subjects. The surface of the plate was painted with black non-‐toxic chalkboard
paint (Rust-‐oleum, Vernon Hills, IL), thereby dividing the plate into four painted quadrants separated by an unpainted area in the shape of a cross. This plate aided observation and quantification of vaginal marking behavior by elevating the subject out of the bedding material; in addition, the divisions on the plate provided a means for quantifying locomotor activity during scent-‐marking tests.
Testing protocol
During each ten-‐minute scent-‐marking test, females were placed within a soiled stimulus cage and the number of vaginal and flank marks were scored using a hand counter by a researcher blind to experimental condition of the subjects. Vaginal marking and flank marking are discrete, stereotyped be-‐ haviors that are differentially expressed in response to conspecific odors (Johnston, 1977). In contrast to vaginal marking, however, flank marking occurs more frequently in response to female than to male odors, and appears to function predominantly in territorial advertisement (Johnston, 1985). A flank mark was scored each time the female moved forward while maintaining contact between the flank re-‐ gion and the side of the stimulus cage. A vaginal mark was scored each time the female moved forward with tail deflected upwards while maintaining contact between the perineum and the underlying sub-‐ strate (Been et al., 2012). Tests were also recorded using a digital camcorder and videos were scored for the number of quadrant entries by researchers blind to the experimental conditions of the subjects, with an inter-‐rater reliability of 90% or greater. Entry into a quadrant was scored whenever greater than 50% of the body mass of the subject crossed from one quadrant into another. At the completion of each scent-‐marking test, the female was removed from the stimulus cage and returned to its home cage. Stimulus cages were not reused; rather, only one female was tested per stimulus cage.
Odor investigation tests
Stimuli
Stimuli for odor investigation tests were collected from the cages of group-‐housed same-‐sex stimulus animals by researchers blind to the experimental condition of the subjects. During collection, approximately 50 ml of soiled corncob bedding and 12 g of soiled cotton nesting material were placed into a one-‐quart re-‐sealable plastic collection bag. In addition, a total of three separate damp gauze pads (10 x 10 cm) were used to wipe down the walls of the cage, the anogenital region, and the bilateral flank glands of odor donors, and these pads were included in the collection bag. Vaginal secretion from odor donor females was collected onto an additional gauze pad by gently palpating the vaginal area with a disposable probe, and included in female odor stimuli collection bags. Hamsters investigate female odors collected on different days of the estrous cycle at relatively equivalent levels (Johnston, 1980); therefore, female stimuli (vaginal secretion, cage stimuli, and body odors) were collected irrespective of cycle day of odor donors. Clean odor stimuli consisted of 10 ml of clean corncob bedding, four grams of clean cotton nesting material, and one clean cotton gauze pad. Once collected, all odor stimuli were stored at 4°C until 30 minutes prior to use.
Apparatus and testing protocol
Odor investigation tests were conducted in a three-‐choice odor investigation apparatus by a re-‐ searcher blind to the experimental condition of the subjects. This apparatus consisted of a modified 51 cm x 25.5 cm x 30.5 cm glass aquarium with opaque paper lining the exterior of the four vertical glass walls and the glass floor. A black line drawn parallel to the short axis of the apparatus bisected the avail-‐ able floor space, allowing activity levels to be quantified. Three 8 cm square acrylic odor containers were attached along the inside of one short wall of the apparatus. Each odor container had a perforated door to allow subjects to investigate the volatile components of the stimuli without allowing direct access to
the contents of the container. The top of the apparatus was secured with a clear Plexiglas lid, allowing for overhead video recording of the subject throughout the behavioral test.
Subjects were tested on two separate occasions in the three-‐choice odor investigation appa-‐ ratus (Clean test and Preference test). Testing occurred across two consecutive estrous cycles, with each test occurring on the cycle day of estrus. Following recovery from stereotaxic surgery, subjects were first tested with clean odor stimuli in each of the three odor containers. This test served to habituate the subjects to the testing protocol. Subjects were then tested in the Preference test. During this test, one of the two outer odor containers contained male odor stimuli, the other outer container contained fe-‐ male odor stimuli, and the center container contained clean odor stimuli. This pattern of odor container placement was designed to maximize the discriminability of male and female odors; however, given that the position of the box containing clean odor stimuli was not alternated during testing, investigatory behavior towards the center box was not included in behavioral analyses for the Preference test.
Subjects were allowed to freely explore the apparatus for ten minutes, and upon completion of the test, subjects were removed, returned to their home cages, and the apparatus was cleaned thor-‐ oughly with 70% ethanol. Odor containers were emptied and cleaned with 70% ethanol. Prior to testing with another subject, fresh odor stimuli were added to the odor containers and the containers were re-‐ placed within the apparatus. The left/right positioning of the male and female odor stimuli containers was counterbalanced across subjects.
All behavior tests were recorded using a digital camcorder and videos from each subject were then scored using the Observer for Windows, version 9.0 (Noldus Information Technology B.V., Wa-‐ geningen, the Netherlands). Researchers blind to the experimental conditions of the subjects scored the videos, with an inter-‐rater reliability of at least 90%. Within each test, the number of midline crosses and the duration of investigation of each of the three odor containers were scored. Investigation was scored